Development of human monoclonal antibodies and uses thereof

ABSTRACT

The present invention provides a trioma cell which does not produce any antibody obtained by fusing a hetermomyeloma cell which does not produce any antibody with a human lymphoid cell, wherein the heteromyeloma cell is designated B6B11. The invention also provides a tetroma cell capable of producing a monoclonal antibody having specific binding affinity for an antigen obtained by fusing a trioma cell which does not produce any antibody with a human lymphoid cell capable of producing antibody having specific binding affinity for the antigen. The invention also provides methods for generating trioma cells and tetroma cells, and the cells generated by the methods.

Throughout this application, various publications are referenced byauthor and date. Full citations for these publications may be foundlisted alphabetically at the end of the specification immediatelypreceding the claims. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art.

BACKGROUND OF THE INVENTION

The seminal discovery by Kohler and Milstein (Kohler, G. and Milstein,C., 1975) of mouse “hybridomas” capable of secreting specific monoclonalantibodies (mAbs) against predefined antigens ushered in a new era inexperimental immunology. Many problems associated with antisera werecircumvented. Clonal selection and immortality of hybridoma cell linesassured monoclonality and permanent availability of antibody products.At the clinical level, however, the use of such antibodies is clearlylimited by the fact that they are foreign proteins and act as antigensto humans.

Since the report of Kohler and Milstein (Kohler, G. and Milstein, C.,1975), the production of mouse monoclonal antibodies has become routine.However, the application of xenogenic mAbs for in vivo diagnostics andtherapy is often associated with undesirable effects such as a humananti-mouse immunoglobulin response. mAbs have great potential as toolsfor imaging; therapeutic treatment has motivated the search into themeans of production of human mAbs (humAbs) (Levy, R., and Miller R A.,1983). However, progress in this area has been hampered by the absenceof human myelomas suitable as fusion partners with the characteristicssimilar to those of mouse myeloma cells (Posner M R, et al., 1983). Theuse of Epstein-Barr virus (EBV) has proved to be quite efficient forhuman lymphocyte immortalization (Kozbor D, and Roder J., 1981; CasualO, 1986), but has certain limitations such as low antibody secretionrate, poor clonogenicity of antibody-secreting lines and chromosomalinstability requiring frequent subcloning. Undifferentiated humanlymphoblastoid cell lines appear more attractive. In contrast todifferentiated myeloma cells, these cell lines are readily adapted toculture conditions, though the problems of low yield and unstablesecretion remain unresolved (Glassy M C, 1983; Ollson L, et al., 1983).The best potential fusion partners are syngenic myeloma cells withwell-developed protein synthesis machinery (Nilsson K. and Ponten J.,1975). However, culturing difficulties explain why few lines have beenconditioned for in vitro growth and capability to produce viable hybrids(Goldman-Leikin R E, 1989). Existing myelomas have low fusion yield andslow hybrid growth, although mAb production is relatively stable (BrodinT, 1983). Genetic instability is a major disadvantage of interspecieshybrids. This is the case, for example, when a mouse myeloma is used asthe immortalizing partner. Production of mouse-human cell hybrids is notdifficult. In vitro these cells have growth characteristics similar tothose of conventional mouse-mouse hybridomas (Teng N N H, 1983).However, spontaneous elimination of human chromosomes considerablyreduces the probability of stable mAb secretion (Weiss M C, and GreenH., 1967). In order to improve growth characteristics and stability ofhumAb production, heterohybrids between mouse myeloma cells and humanlymphocyte (Oestberg L, and Pursch E., 1983) as well as heteromyelomas(Kozbor D, et. al., 1984) are used as the fusion partners.

SUMMARY OF THE INVENTION

The present invention provides a heteromyeloma cell which does notproduce any antibody, capable of producing a trioma cell which does notproduce any antibody, when fused with a human lymphoid cell, wherein thetrioma cell is capable of producing a tetroma cell capable of producinga monoclonal antibody having specific binding affinity for an antigen,when fused with a second human lymphoid cell, the second human lymphoidcell being capable of producing antibody having specific bindingaffinity for the antigen, with the proviso that the heteromyeloma cellis not B6B11 (ATCC Designation Number HB-12481).

The present invention further provides a trioma cell obtained by fusinga heteromyeloma cell which does not produce any antibody with a humanlymphoid cell.

The present invention also provides a tetroma cell capable of producinga monoclonal antibody having specific binding affinity for an antigen,obtained by fusing the described trioma cell which does not produce anyantibody with a human lymphoid cell capable of producing antibody havingspecific binding affinity for the antigen.

The present invention additionally provides a monoclonal antibodyproduced by the described tetroma.

The present invention further provides a method of generating thedescribed trioma cell comprising: (a) fusing a heteromyeloma cell whichdoes not produce any antibody with a human lymphoid cell thereby forminga trioma cell; (b) incubating the trioma cell formed in step (a) underconditions permissive to the production of antibody by the trioma cell;and (c) selecting a trioma fusion cell that does not produce anyantibody.

Still further, the present invention provides a method of generating atetroma cell comprising: (a) fusing the described trioma cell with ahuman lymphoid cell, thereby forming a tetroma cell; (b) incubating thetetroma cell formed in step (a) under conditions permissive to theproduction of antibody by the tetroma cell; and (c) selecting a tetromacell capable of producing a monoclonal antibody.

The present invention also provides a method of producing a monoclonalantibody comprising (a) fusing a lymphoid cell capable of producing ofproducing antibody with the described trioma cell, thereby forming atetroma cell; and (b) incubating the tetroma cell formed in step (a)under conditions permissive to the production of antibody by the tetromacell, thereby producing the monoclonal antibody.

Also the present invention provides a method of producing a monoclonalantibody specific for an antigen associated with a condition in asubject comprising: (a) fusing a lymphoid cell capable of producingantibody with the described trioma cell, thereby forming a tetroma cell;(b) incubating the tetroma cell formed in step (a) under conditionspermissive to the production of antibody by the tetroma cell; (c)selecting a tetroma cell producing a monoclonal antibody; (d) contactingthe monoclonal antibody of step (c) with (1) a sample from a subjectwith the condition or (2) a sample from a subject without the conditionunder conditions permissive to the formation of a complex between themonoclonal antibody and the sample; (e) detecting the complex formedbetween the monoclonal antibody and the sample; (f) determining theamount of complex formed in step (e); and (g) comparing the amount ofcomplex determined in step (f) for the sample from the subject with thecondition with amount determined in step (f) for the sample from thesubject without the condition, a greater amount of complex formation forthe sample from the subject with the condition indicating that amonoclonal antibody specific for the antigen specific for the conditionis produced.

Additionally, the present invention provides a method of identifying anantigen associated with a condition in a sample comprising: (a)contacting the monoclonal antibody produced by the described method withthe sample under conditions permissive to the formation of a complexbetween the monoclonal antibody and the sample; (b) detecting thecomplex formed in step (a); and (c) isolating the complex detected instep (b), thereby identifying the antigen associated with the conditionin the sample.

The present invention additionally provides a method of diagnosing acondition in a subject comprising: (a) contacting a sample from thesubject with a monoclonal antibody produced by the described methodunder conditions permissive to the formation of a complex between themonoclonal antibody and the sample; and (b) detecting the formation of acomplex between the monoclonal antibody and the sample, positivedetection indicating the presence of an antigen specific for thecondition in the sample, thereby diagnosing the condition in thesubject.

The present invention further provides a composition comprising theproduced monoclonal antibody and a suitable carrier.

Further, the present invention also provides a therapeutic compositioncomprising an effective amount of the produced monoclonal antibody and apharmaceutically acceptable carrier.

Also, the present invention further provides a method of treating acondition in a subject comprising administering to the subject an amountof the described therapeutic composition effective to bind the antigenassociated with the condition, thereby treating the condition in thesubject.

Finally, the present invention provides a method of preventing acondition in a subject comprising administering to the subject an amountof the described therapeutic composition, effective to bind the antigenassociated with the condition, thereby preventing the condition in thesubject.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C Distribution of cells according to the number ofchromosomes. The X-axis indicates the amount of chromosomes. The Y-axisindicates the percentage of cells with appropriate number ofchromosomes. The data represent the average ones based on the analysisof more than 50 metaphase plates for each line: P3.X63.Ag8.653 FIG. 1A,RPMI 8226 FIG. 1B, B6B11 FIG. 1C.

FIG. 2 Fragment of G-banded karyotype of B6B11 line. The arrows indicategenetic material presumably of human origin; 3p portion of chromosome 3and chromosome 19.

FIG. 3 B6B11 fusion efficiency with fresh isolated and culturedsplenocytes. SPL were isolated in LSM, immediately after a portion ofthe cells were fused with B6B11 cells and the remaining SPL werecultivated in vitro for 7-9 days in RPMI-C containing 15% FCS in thepresence of ConA, LPS, PHA, PWM or without mitogens, then these cellswere also fused with B6B11. PWM in the concentration of 5 μg/mlinfluenced effectively the fusion efficiency.

FIGS. 4A-4D DNA histograms of parental cells 653 (FIG. 4A) and 8226(FIG. 4B), heteromyeloma B6B11 (FIG. 4C) and B6B11-splenocyte hybrid(FIG. 4D). The amount of B6B11 DNA constitutes about 100% of the totalamount of 653 DNA plus 8226 DNA. The DNA content of B6B11-SPL hybrid isgreater than that of B6B11.

FIGS. 5A-5B Immunoglobulin production by hybridomas (tetromas) derivedfrom the fusion of PBLs with MFP-2. FIG. 5A shows results of fusingfresh lymphocyte suspensions with MFP-2. FIG. 5B shows results of fusingfrozen/thawed lymphocyte suspensions with MFP-2. The dark rectanglesindicate IgM production. The gray rectangles indicate IgG production.The Y-axis indicates optical density at A₄₉₀ for different hybridomasamples (tetromas) generated from fusion with the MFP-2 trioma line(X-axis). The dotted line indicates the optical density at A₄₉₀ for a1:500 dilution of IgM antibody. The dashed line indicates the opticaldensity at A₄₉₀ for a 1:500 dilution of IgG antibody.

FIG. 6 Anti-thyroglobulin antibody production by thyroid cancer lymphnode lymphocytes fused to fusion partner MFP-2 cells. The Y-axisindicates optical density at A₄₀₅ (OD₄₀₅) for different hybridomasamples (tetromas) generated from fusion with the MFP-2 trioma line(X-axis). Thirty-three tetromas produced antibody which reactedpositively against thyroglobulin; eight were particularly stronglyreactive.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a heteromyeloma cell which does notproduce any antibody, capable of producing a trioma cell which does notproduce any antibody, when fused with a human lymphoid cell, wherein thetrioma cell is capable of producing a tetroma cell capable of producinga monoclonal antibody having specific binding affinity for an antigen,when fused with a second human lymphoid cell, the second human lymphoidcell being capable of producing antibody having specific bindingaffinity for the antigen, with the proviso that the heteromyeloma cellis not B6B11 (ATCC Designation number HB-12481). Heteromyeloma cellB6B11 was deposited on Mar. 17, 1998 with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va., U.S.A.under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganism for the Purposes of PatentProcedure. B6B11 was accorded ATCC Designation Number HB-12481.

The present invention provides a trioma cell obtained by fusing aheteromyeloma cell which does not produce any antibody with a humanlymphoid cell. In an embodiment of this invention, the heteromyelomacell is designated B6B11 (ATCC Designation number HB-12481). In anotherembodiment, the trioma is a B6B11-like cell. In an embodiment of thisinvention, the human lymphoid cell is a myeloma cell. In anotherembodiment of this invention, the human lymphoid cell is a splenocyte ora lymph node cell (lymphocyte). According to an embodiment of thisinvention, the trioma cell is designated MFP-2 (ATCC Designation numberHB-12482). Trioma cell MFP-2 was deposited on Mar. 17, 1998 with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va., U.S.A. under the provisions of the Budapest Treaty forthe International Recognition of the Deposit of Microorganism for thePurposes of Patent Procedure. MFP-2 was accorded ATCC Designation NumberHB-12482.

The present invention also provides a tetroma cell capable of producinga monoclonal antibody having specific binding affinity for an antigen,obtained by fusing the described trioma cell which does not produce anyantibody with a human lymphoid cell capable of producing antibody havingspecific binding affinity for the antigen. According to an embodiment ofthis invention, the human lymphoid cell is a peripheral bloodlymphocyte, a splenocyte, a lymph node cell, a B cell, a T cell, atonsil gland lymphocyte, a monocyte, a macrophage, an erythroblastoidcell or a Peyer's patch cell. In an embodiment of this invention, thetrioma cell is designated MFP-2 (ATCC Designation number HB-12482).

According to an embodiment of this invention, the antigen is atumor-associated antigen, a cell-specific antigen, a tissue-specificantigen, an enzyme, a nucleic acid, an immunoglobulin, a toxin, a viralantigen, a bacterial antigen or a eukaryotic antigen. In one embodiment,the antigen is a mammalian, insect, fungal, E. coli or Klebsiellaantigen.

The present invention provides a monoclonal antibody produced by thedescribed tetroma. The present invention also provides an isolatednucleic acid encoding the monoclonal antibody produced by the describedtetroma. The nucleic acid may include, but is not limited to DNA, RNA,cDNA, oligonucleotide analogs, vectors, expression vectors or probes.Additionally, the present invention contemplates the expression of thenucleic acid encoding the monoclonal antibody introduced into a hostcell capable of expression the monoclonal antibody or portions thereof.

The present invention further provides a method of generating thedescribed trioma cell comprising: (a) fusing a heteromyeloma cell whichdoes not produce any antibody with a human lymphoid cell thereby forminga trioma cell; (b) incubating the trioma cell formed in step (a) underconditions permissive to the production of antibody by the trioma cell;and (c) selecting a trioma fusion cell that does not produce anyantibody.

According to one embodiment of this invention, the heteromyeloma cell ofstep (a) is designated B6B11 (ATCC Designation number HB-12481).According to an embodiment of this invention, the human lymphoid cell isa lymph node lymphocyte or a splenocyte. According to an embodiment ofthe present invention, the method further comprises selecting a triomacell capable of growth in serum-free media. Another embodiment furthercomprises selecting a trioma cell that is capable of fusing with aperipheral blood lymphocyte or lymph node lymphocyte. The presentinvention provides a trioma cell generated by the described method.

Still further, the present invention provides a method of generating atetroma cell comprising: (a) fusing the described trioma cell with ahuman lymphoid cell thereby forming a tetroma cell; (b) incubating thetetroma cell formed in step (a) under conditions permissive to theproduction of antibody by the tetroma cell; and (c) selecting a tetromacell capable of producing a monoclonal antibody. According to oneembodiment of this invention, the trioma cell of step (a) is designatedMFP-2 (ATCC Designation number HB-12482). According to an embodiment ofthis invention, the human lymphoid cell is a peripheral bloodlymphocyte, a splenocyte, a lymph node cell, a B cell, a T cell, atonsil gland lymphocyte, a monocyte, a macrophage, an erythroblastoidcell or a Peyer's patch cell. In an embodiment of this invention, thehuman lymphoid cell produces antibodies having specific binding affinityfor an antigen and wherein the tetroma cell produces a monoclonalantibody having specific binding affinity for the antigen. According toan embodiment of this invention, the antigen is a tumor-associatedantigen, a cell-specific antigen, a tissue-specific antigen, an enzyme,a nucleic acid, an immunoglobulin, a toxin, a viral antigen, a bacterialantigen or a eukaryotic antigen. In an embodiment of this invention, theantigen is a mammalian, insect, E. coli or Klebsiella antigen. Thepresent invention further provides a tetroma cell generated by thedescribed method.

The present invention also provides a method of producing a monoclonalantibody comprising (a) fusing a lymphoid cell capable of producingantibody with the described trioma cell, thereby forming a tetroma cell;and (b) incubating the tetroma cell formed in step (a) under conditionspermissive to the production of antibody by the tetroma cell, therebyproducing the monoclonal antibody.

Also, the present invention provides a method of producing a monoclonalantibody specific for an antigen associated with a condition in asubject comprising: (a) fusing a lymphoid cell capable of producingantibody with the described trioma cell, thereby forming a tetroma cell;(b) incubating the tetroma cell formed in step (a) under conditionspermissive to the production of antibody by the tetroma cell; (c)selecting a tetroma cell producing a monoclonal antibody; (d) contactingthe monoclonal antibody of step (c) with (1) a sample from a subjectwith the condition or (2) a sample from a subject without the conditionunder conditions permissive to the formation of a complex between themonoclonal antibody and the sample; (e) detecting the complex formedbetween the monoclonal antibody and the sample; (f) determining theamount of complex formed in step (e); and (g) comparing the amount ofcomplex determined in step (f) for the sample from the subject with thecondition with amount determined in step (f) for the sample from thesubject without the condition, a greater amount of complex formation forthe sample from the subject with the condition indicating that amonoclonal antibody specific for the antigen specific for the conditionis produced.

In one embodiment of the present invention, step (a) further comprisesfreezing the lymphoid cell. According to one embodiment of the presentinvention, step (c) further comprises incubating the selected tetromacell under conditions permissive to cell replication. According to anembodiment of this invention, the tetroma replication is effected invitro or in vivo. According to one embodiment of this invention, thetrioma cell is designated MFP-2 (ATCC Designation No. HB-12482). Thepresent invention provides a monoclonal antibody specific for an antigenassociated with a condition, identified by the described method. Thepresent invention also provides an isolated nucleic acid encoding thedescribed monoclonal antibody. The nucleic acid may include, but is notlimited to DNA, RNA, cDNA, oligonucleotide analogs, vectors, expressionvectors or probes. Additionally, the present invention contemplates theexpression of the nucleic acid encoding the monoclonal antibodyintroduced into a host cell capable of expression the monoclonalantibody or portions thereof.

According to an embodiment of this invention, the condition isassociated with a cancer, a tumor, a toxin, an infectious agent, anenzyme dysfunction, a hormone dysfunction, an autoimmune disease, animmune dysfunction, a viral antigen, a bacterial antigen, a eukaryoticantigen, rejection of a transplanted tissue, poisoning, or venomintoxication. Additionally the condition may be any other abnormality,including that resulting from infection, cancer, autoimmune dysfunction,cardiovascular disease, or transplantation. In an embodiment of thisinvention, the condition is septicemia, sepsis, septic shock, viremia,bacteremia or fungemia. In an embodiment of this invention, the canceris lung cancer, liver cancer, leukemia, lymphoma, neuroblastoma, glioma,meningioma, bone cancer, thyroid cancer, breast cancer or prostatecancer. According to an embodiment of this invention, the infectiousagent is Hanta virus, HTLV I, HTLV II, HIV, herpes virus, influenzavirus, Ebola virus, human papilloma virus, Staphlococcus, Streptococcus,Klebsiella, E. coli, anthrax or cryptococcus. According to an embodimentof this invention, the toxin is tetanus, anthrax, botulinum snake venomor spider venom. In one embodiment of this invention, the tumor isbenign. In another embodiment, the enzyme dysfunction is hyperactivityor overproduction of the enzyme. In still another embodiment, thehormone dysfunction is hyperactivity or overproduction of the hormone.In yet another embodiment of this invention, the immune dysfunction isCD3 or CD4 mediated. In still another embodiment of this invention, theautoimmune disease is lupus, thyroidosis, graft versus host disease,transplantation rejection or rheumatoid arthritis. In still anotherembodiment of the invention, the condition is any abnormality. In stillanother embodiment, the condition is the normal condition.

Additionally, the present invention provides a method of identifying anantigen associated with a condition in a sample comprising: (a)contacting the monoclonal antibody produced by the described method withthe sample under conditions permissive to the formation of a complexbetween the monoclonal antibody and the sample; (b) detecting thecomplex formed in step (a); and (c) isolating the complex detected instep (b), thereby identifying the antigen associated with the conditionin the sample.

An embodiment of this invention, further comprises separating themonoclonal antibody from the monoclonal antibody-antigen complex. Inanother embodiment the separation is by size fractionation. According toone embodiment, the size fractionation is effected by polyacrylamide oragarose gel electrophoresis.

According to an embodiment of this invention, the condition isassociated with a cancer, a tumor, a toxin, an infectious agent, anenzyme dysfunction, a hormone dysfunction, an autoimmune disease, animmune dysfunction, a viral antigen, a bacterial antigen, a eukaryoticantigen, rejection of a transplanted tissue, poisoning, or venomintoxication. Additionally the condition may be any other abnormality,including that resulting from infection, cancer, autoimmune dysfunction,cardiovascular disease, or transplantation. In an embodiment of thisinvention, the condition is septicemia, sepsis, septic shock, viremia,bacteremia or fungemia. In an embodiment of this invention, the canceris lung cancer, liver cancer, leukemia, lymphoma, neuroblastoma, glioma,meningioma, bone cancer, thyroid cancer, breast cancer or prostatecancer. According to an embodiment of this invention, the infectiousagent is Hanta virus, HTLV I, HTLV II, HIV, herpes virus, influenzavirus, Ebola virus, human papilloma virus, Staphlococcus, Streptococcus,Klebsiella, E. coli, anthrax or cryptococcus. According to an embodimentof this invention, the toxin is tetanus, anthrax, botulinum, snake venomor spider venom. In one embodiment of this invention, the tumor isbenign. In another embodiment, the enzyme dysfunction is hyperactivityor overproduction of the enzyme. In still another embodiment, thehormone dysfunction is hyperactivity or overproduction of the hormone.In yet another embodiment of this invention, the immune dysfunction isCD3 or CD4 mediated. In still another embodiment of this invention, theautoimmune disease is lupus, thyroidosis, graft versus host disease,transplantation rejection or rheumatoid arthritis. In still anotherembodiment of the invention, the condition is any abnormality. In stillanother embodiment, the condition is the normal condition.

The present invention additionally provides a method of diagnosing acondition in a subject comprising: (a) contacting a sample from thesubject with a monoclonal antibody produced by the described methodunder conditions permissive to the formation of a complex between themonoclonal antibody and the sample; and (b) detecting the formation of acomplex between the monoclonal antibody and the sample, positivedetection indicating the presence of an antigen specific for thecondition in the sample, thereby diagnosing the condition in thesubject.

According to an embodiment of this invention, the monoclonal antibody iscoupled to a detectable marker. In an embodiment of this invention, thedetectable marker is a radiolabeled molecule, a fluorescent molecule, anenzyme, a ligand, a colorimetric marker or a magnetic bead.

According to an embodiment of this invention, the condition isassociated with a cancer, a tumor, a toxin, an infectious agent, anenzyme dysfunction, a hormone dysfunction, an autoimmune disease, animmune dysfunction, a viral antigen, a bacterial antigen, a eukaryoticantigen, rejection of a transplanted tissue, poisoning, or venomintoxication. Additionally the condition may be any other abnormality,including that resulting from infection, cancer, autoimmune dysfunction,cardiovascular disease, or transplantation. In an embodiment of thisinvention, the condition is septicemia, sepsis, septic shock, viremia,bacteremia or fungemia. In an embodiment of this invention, the canceris lung cancer, liver cancer, leukemia, lymphoma, neuroblastoma, glioma,meningioma, bone cancer, thyroid cancer, breast cancer or prostatecancer. According to an embodiment of this invention, the infectiousagent is Hanta virus, HTLV I, HTLV II, HIV, herpes virus, influenzavirus, Ebola virus, human papilloma virus, Staphlococcus, Streptococcus,Klebsiella, E. coli, anthrax or cryptococcus. According to an embodimentof this invention, the toxin is tetanus, anthrax, botulinum, snake venomor spider venom. In one embodiment of this invention, the tumor isbenign. In another embodiment, the enzyme dysfunction is hyperactivityor overproduction of the enzyme. In still another embodiment, thehormone dysfunction is hyperactivity or overproduction of the hormone.In yet another embodiment of this invention, the immune dysfunction isCD3 or CD4 mediated. In still another embodiment of this invention, theautoimmune disease is lupus, thyroidosis, graft versus host disease,transplantation rejection or rheumatoid arthritis. In still anotherembodiment of the invention, the condition is any abnormality. In stillanother embodiment, the condition is the normal condition.

The present invention further provides a composition comprising theproduced monoclonal antibody and a suitable carrier.

Further, the present invention also provides a therapeutic compositioncomprising an effective amount of the produced monoclonal antibody and apharmaceutically acceptable carrier.

According to an embodiment of this invention, the condition is cancerand the amount of monoclonal antibody is sufficient to inhibit thegrowth of or eliminate the cancer. According to an embodiment, thecondition is an infection and the amount of monoclonal antibody issufficient to inhibit the growth of or kill the infectious agent.According to an embodiment of this invention, the condition is associatewith a toxin and the amount of monoclonal antibody is sufficient toreduce the amount of or destroy the toxin. In still another embodiment,the condition is an autoimmune disease and the amount of monoclonalantibody is sufficient to reduce the amount of or destroy the offendingantibody or subunit(s) thereof. In still another embodiment, thecondition is a cardiovascular disease and the amount of monoclonalantibody is sufficient to reduce the condition. In yet anotherembodiment, the condition is a transplantation rejection, and the amountof monoclonal antibody is sufficient to reduce the condition.

According to an embodiment of this invention, the monoclonal antibody iscoupled to an effector compound. In an embodiment of this invention, theeffector compound is a cytotoxic agent, drug, enzyme, dye, orradioisotope. In an embodiment of this invention, the monoclonalantibody is coupled to a carrier. According to one embodiment of thisinvention, the carrier is a liposome.

Also, the present invention further provides a method of treating acondition in a subject comprising administering to the subject an amountof the described therapeutic composition effective to bind the antigenassociated with the condition, thereby treating the condition in thesubject. According to one embodiment of this invention, the therapeuticcomposition is administered to a second subject.

According to an embodiment of this invention, the condition isassociated with a cancer, a tumor, a toxin, an infectious agent, anenzyme dysfunction, a hormone dysfunction, an autoimmune disease, animmune dysfunction, a viral antigen, a bacterial antigen, a eukaryoticantigen, rejection of a transplanted tissue, poisoning, or venomintoxication. Additionally the condition may be any other abnormality,including that resulting from infection, cancer, autoimmune dysfunction,cardiovascular disease, or transplantation. In an embodiment of thisinvention, the condition is septicemia, sepsis, septic shock, viremia,bacteremia or fungemia. In an embodiment of this invention, the canceris lung cancer, liver cancer, leukemia, lymphoma, neuroblastoma, glioma,meningioma, bone cancer, thyroid cancer, breast cancer or prostatecancer. According to an embodiment of this invention, the infectiousagent is Hanta virus, HTLV I, HTLV II, HIV, herpes virus, influenzavirus, Ebola virus, human papilloma virus, Staphlococcus, Streptococcus,Klebsiella, E. coli, anthrax or cryptococcus. According to an embodimentof this invention, the toxin is tetanus, anthrax, botulinum, snake venomor spider venom. In one embodiment of this invention, the tumor isbenign. In another embodiment, the enzyme dysfunction is hyperactivityor overproduction of the enzyme. In still another embodiment, thehormone dysfunction is hyperactivity or overproduction of the hormone.In yet another embodiment of this invention, the immune dysfunction isCD3 or CD4 mediated. In still another embodiment of this invention, theautoimmune disease is lupus, thyroidosis, graft versus host disease,transplantation rejection or rheumatoid arthritis. In still anotherembodiment of the invention, the condition is any abnormality. In stillanother embodiment, the condition is the normal condition.

Finally, the present invention provides a method of preventing acondition in a subject comprising administering to the subject an amountof the described therapeutic composition, effective to bind the antigenassociated with the condition, thereby preventing the condition in thesubject. In one embodiment of this invention, the subject previouslyexhibited the condition. According to one embodiment of this invention,the therapeutic composition is administered to a second subject.

According to an embodiment of this invention, the condition isassociated with a cancer, a tumor, a toxin, an infectious agent, anenzyme dysfunction, a hormone dysfunction, an autoimmune disease, animmune dysfunction, a viral antigen, a bacterial antigen, a eukaryoticantigen, rejection of a transplanted tissue, poisoning, or venomintoxication. Additionally the condition may be any other abnormality,including that resulting from infection, cancer, autoimmune dysfunction,cardiovascular disease, or transplantation. In an embodiment of thisinvention, the condition is septicemia, sepsis, septic shock, viremia,bacteremia or fungemia. In an embodiment of this invention, the canceris lung cancer, liver cancer, leukemia, lymphoma, neuroblastoma, glioma,meningioma, bone cancer, thyroid cancer, breast cancer or prostatecancer. According to an embodiment of this invention, the infectiousagent is Hanta virus, HTLV I, HTLV II, HIV, herpes virus, influenzavirus, Ebola virus, human papilloma virus, Staphlococcus, Streptococcus,Klebsiella, E. coli, anthrax or cryptococcus. According to an embodimentof this invention, the toxin is tetanus, anthrax, botulinum, snake venomor spider venom. In one embodiment of this invention, the tumor isbenign. In another embodiment, the enzyme dysfunction is hyperactivityor overproduction of the enzyme. In still another embodiment, thehormone dysfunction is hyperactivity or overproduction of the hormone.In yet another embodiment of this invention, the immune dysfunction isCD3 or CD4 mediated. In still another embodiment of this invention, theautoimmune disease is lupus, thyroidosis, graft versus host disease,transplantation rejection or rheumatoid arthritis. In still anotherembodiment of the invention, the condition is any abnormality. In stillanother embodiment, the condition is the normal condition.

The present invention also provides the production of antibodies forantigens which are not associated with a condition, but more properlyconstitute a component of the entire repetoire of antibodies in a humanimmune system.

In addition, the present invention provides identification of novelantigens relevant to a condition in a subject and the use thereof fordiagnosis and treatment of the condition in the subject. The inventionalso provides identification of the repetoire of naturally occurringantibodies in normal subjects and subjects having a pathologicalcondition. In one embodiment, the condition may be venomdetoxicification (neutralization). For example, the condition may resultfrom scorpion, spider, rattle snake or poison toad bites or venomexposure. The present invention provides antibodies to act as antidotefor such conditions.

The trioma cell of the present invention may also be fused withmacrophages, monocytes, T-lymphocytes, and erythroblastoid cells.Hybridoma cells resulting from such fusions may produce growth factors,cytokines, enzymes, hemoglobin

As used herein, a human-murine hybridoma (the “immortalizing hybridoma”)is an immortal cell line which results from the fusion of a murinemyeloma or other murine tumor cell with a human lymphoid cell derivedfrom a normal subject. As described herein below, by careful selectionand mutation, an immortalizing hybridoma which provides improvedchromosomal stability, has human characteristics, and which does notsecrete immunoglobulin may be obtained. The antibody secretingcapability of such a resulting trioma may be provided by the third cellfusion which is typically derived either from B cells of an immunizedhuman individual, or with B cells which have been immortalized.

As used herein, a “B6B11” cell is a hybrid cell produced by the fusionof mouse myeloma 653 and human myeloma RPMI 8226.

As used herein, a “B6B11-like” cell is a a hybrid cell produced by thefusion of mouse myeloma 653-related cell and human myeloma RPMI8226-related cell.

As used herein, a “MFP” cell is a hybrid cell produced by the fusion ofa B6B11 cell and a human lymphocyte. B6B11-like cells share functionproperties and characteristics with B6B11 heteromyeloma cells.

As used herein, a “MFP-like” cell is a hybrid cell produced by thefusion of a B6B11-like cell and a human lymphocyte. MFP-like cells sharefunction properties and characteristics with MFP trioma cells.

As used herein, “non-secreting” or “non-producing” hybridoma refers to ahybridoma which is capable of continuous reproduction and, therefore, isimmortal, and which does not produce immunoglobulin.

As used herein, a hybridoma “having human characteristics” refers to ahybridoma which retains detectable human-derived chromosomes such asthose producing human HLA antigen which may be expressed on the cellsurface.

As used herein, lymphoid cells “immunized against a predefineddeterminant” refers to lymphoid cells derived from an subject who hasbeen exposed to an antigen having the determinant. For example, asubject can be induced to produce (from its lymphoid B cells) antibodiesagainst the antigenic determinants of various blood types, by exposure,through transfusions or previous pregnancy, or against the antigenicdeterminants of specific viruses or of bacteria by virus of exposurethrough past infections or vaccinations.

As used herein, “cell line” refers to various embodiments including butnot limited to individual cells, harvested cells and cultures containingcells so long as these are derived from cells of the cell line referredto may not be precisely identical to the ancestral cells or cultures andany cell line referred to include such variants.

As used herein, “trioma” refers to a cell line which contains genericcomponents originating in three originally separate cell linages. Thesetriomas are stable, immortalized cells which result from the fusion of ahuman-murine hybridoma with a human lymphoid cell.

As used herein, “tetroma” refers to a a cell line which contains genericcomponents originating in four originally separate cell lineages. Thesetetromas are stable, immortalized antibody producing cells which resultfrom the fusion of a trioma with a human lymphoid cell which is capableof producing antibody.

As used herein, “autologously” refers to a situation where the samesubject is both the source of cell immunoglobulin and the target forcells, or immunoglobulin or therapeutic composition.

As used herein, “heterologously” refers to a situation where one subjectis the source of cells or immunoglobulin and another subject is thetarget for the cell, immunoglobulin or therapeutic composition.

In the practice of any of the methods of the invention or preparation ofany of the pharmaceutical compositions a “therapeutically effectiveamount” is an amount which is capable of binding to an antigenassociated with the condition. Accordingly, the effective amount willvary with the subject being treated, as well as the condition to betreated. For the purposes of this invention, the methods ofadministration are to include, but are not limited to, administrationcutaneously, subcutaneously, intravenously, parenterally, orally,topically, or by aerosol.

As used herein, the term “suitable pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers, suchas phosphate buffered saline solution, water, emulsions such as anoil/water emulsion or a triglyceride emulsion, various types of wettingagents, tablets, coated tablets and capsules. An example of anacceptable triglyceride emulsion useful in intravenous andintraperitoneal administration of the compounds is the triglycerideemulsion commercially known as Intralipid®.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients.

This invention also provides for pharmaceutical compositions capable ofbinding to an antigen associated with the condition together withsuitable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions are liquids or lyophilized orotherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),solubilizing agents (e.g., glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the compound, complexation withmetal ions, or incorporation of the compound into or onto particulatepreparations of polymeric compounds such as polylactic acid, polglycolicacid, hydrogels, etc, or onto liposomes, micro emulsions, micelles,unilamellar or multi lamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance of the compound or composition.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended bythe invention are particulate compositions coated with polymers (e.g.,poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

When administered, compounds are often cleared rapidly from thecirculation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of bioactive compounds may by required to sustaintherapeutic efficacy. Compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline areknown to exhibit substantially longer half-lives in blood followingintravenous injection than do the corresponding unmodified compounds(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987).Such modifications may also increase the compound's solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound adducts less frequently or in lower doses thanwith the unmodified compound.

Attachment of polyethylene glycol (PEG) to compounds is particularlyuseful because PEG has very low toxicity in mammals (Carpenter et al.,1971). For example, a PEG adduct of adenosine deaminase was approved inthe United States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. A second advantage afforded by theconjugation of PEG is that of effectively reducing the immunogenicityand antigenicity of heterologous compounds. For example, a PEG adduct ofa human protein might be useful for the treatment of disease in othermammalian species without the risk of triggering a severe immuneresponse. The carrier includes a microencapsulation device so as toreduce or prevent an host immune response against the compound oragainst cells which may produce the compound. The compound of thepresent invention may also be delivered microencapsulated in a membrane,such as a liposome.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxy-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

The present invention describes the production of human monoclonalantibodies directed to tumor-associated antigens, tumor cells,infectious agents, infection-specific antigens, and self antigens usinga modified cell fusion partner, trioma cell line and human lymphocytesderived from lymph nodes, spleen, Peyer's patches, or any other lymphtissue or peripheral blood of the human subjects.

Antibodies are selected using cultured cells, purified antigens, primaryhuman cells and tissues and combinatorial libraries relevant to theantibody screening including cells and tissues obtained from autologousdonor of lymphoid cells.

This invention is illustrated by examples set forth in the ExperimentalDetails section which follows. This section is provided to aid in anunderstanding of the invention but is not intended to, and should not beconstrued to, limit in any way the invention as set forth in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS EXAMPLE 1 Construction of Mouse-human Heteromyelomafor the Production of Human Monoclonal antibodies Introduction

B6B11 or B6B11-like cells may be produced by the fusion of mouse myelomacells with human myeloma cells selected for non-secretion of antibody.The specific generation and application of heteromyeloma B6B11, isdescribed herein below. B6B11 was obtained by fusing the mouseHAT-sensitive and G-418 resistant myeloma X63.Ag8.653 with the subcloneof human myeloma RPMI 8226 selected for non secretion of lambda lightchains. Fusion of human splenocytes and B6B11 cells resulted in a fusionfrequency of 30-50 hybrids per 10⁷ cells. This is similar to thefrequency of murine hybridoma formation. The hybrids are readily clonedby limiting dilution, produce antibodies for at least 10 month and growin serum-free media. Two clones were obtained which secreted human IgMreactive against lipopolysaccharide (LPS) of Gram-negative bacteria.These clones were obtained by fusing in vitro immunized humansplenocytes with the B6B11 cells. Anti-lipid A murine mAb is known toprevent development of septic shock (Shnyra A A, et al., 1990). HumanmAbs have important clinical applications.

Results

Heteromyeloma B6B11. Heteromyeloma, B6B11, was generated by PEG-fusionof mouse myeloma 653 (HAT-sensitive, G-418) with human RPMI 8226, whichwas selected for non-secretion of lambda chains. Hybrids were selectedin the presence of HAT and G-418. Selection for 8-Ag resistance was doneby gradually increasing the 8-Ag concentration from 2 ug/ml to 20 ug/mlfor 2.5-3 weeks. The HAT-sensitive hybrid population 653×8226 was twicecloned.

Clones were tested for the ability to produce hybrids with humanlymphocytes. One clone, designated as B6B11, was selected. B6B11 cellsdied in medium containing aminopterine, during a period of 5-6 days; norevertants were detected for more than 18 months. In RPMI 1640supplemented with 10% fetal calf serum (FCS), the line had the doublingtime of about 25-30 hours, the maximal density in 75 cm² flasks wasapproximately 1.5×10⁶ cells/ml (in a volume of 30 ml). B6B11 culturemedium was tested for the presence of human immunoglobulin by enzymelinked immunoassay (ELISA) using rabbit anti-human immunoglobulin. B6B11exhibited secretion of IgG, IgM or IgA. Staining the cell preparationswith MAH-L,H by PAP-technique detected no traces of cytoplasmic lightand heavy chain human immunoglobulin.

Karyotyping. FIG. 1 illustrates the distribution of parental and B6B11cells by chromosomal content. Chromosomal analysis of the heteromyelomacells indicated that chromosomal number varies from 60 to 82.

FIG. 2 shows a fragment of the G-banded karyotype of B6B11 cells. Normalmouse chromosomes constitute about 84% of the karyotype. There areseveral rearranged chromosomes. There are some markers for mouse myelomachromosomes as well as rearranged heteromyeloma (human-mouse chimeric)chromosomes. One large telocentric chromosome was represented in allB6B11 metaphase plates examined. This suggested that the proximalportion of this chromosome contains mouse and the distal portioncontains human genetic material of chromosome 3 (3p21.1-3p ter).Localization of human material was performed as described (33). In someof analyzed B6B11, cells human chromosome 19 and human chromosome 7 wasdeleted.

Fusion Of B6B11 Cells With Human Lymphocytes. Fusion of B6B11 cells withfreshly isolated peripheral blood lymphocytes (PBL) and spleniclymphocytes (SPL) was performed as described herein below in theExperimental Procedures Section. Fusion of peripheral blood lymphocytes(PBL) and pokeweed mitogen (PWM) treated peripheral blood lymphocytes(PBL) resulted in low hybridoma yield (1-5 hybrids per 10⁷ lymphocytes),while fusion with splenic lymphocytes (SPL) and pokeweed mitogen (PWM)treated splenic lymphocytes (SPL) yielded 30-60 hybrids per 10⁷ cells(see Table 1). After the fusion, cells were seeded at a density of1.5×10⁵ cells per well. Variations in the cell ratios of 1:1 to 1:2(heteromyeloma:lymphocyte) had no effect on the fusion efficiency forPBL or SPL. However, fusion efficiency was dramatically reduced atB6B11: lymphocyte ratios of 1:4 to 1:8.

TABLE 1 Fusion of human lymphocytes with B6B11 cells. LYMPHOCYTES PBLPBL-PWM SPL SPL-PWM Number of fusion 4 6 10 8 Number of wells 1536 23044800 3072 Growth², % 4 6, 9 55 72 Hybrid populations³ 1-3 3-5 30-5040-60 per 10⁷ lymphocytes Wells with Ig 95 92 84 82 secretion⁴, % ¹Freshisolated peripheral blood lymphocytes (PBL) and splenocytes (SPL) wereactivated with PWM (5 ug/ml) for 7-9 days in complete RPMI 1640supplemented with 15% FCS. ²Wells with hybrids (% of the total wellnumber) ³After fusion cells were seeded at a density of 15 × 10⁴cells/well ⁴Total Ig production was determined by ELISA with mousemonoclonal antibodies to H- and L-chains of human Ig

The effects of splenocyte stimulation with various mitogens on thefusion efficiency are illustrated in FIG. 3. PWM treatment significantlyincreased the efficiency of SPL hybridization compared withConA-treatment, PHA-treatment, LPS-treatment or untreated SPL. Fusionefficiency was dependent on the timing of the HAT addition. When HAT wasadded immediately following fusion, the yield decreased to 10-15 hybridsper 10⁷ lymphocytes (for SPL).

Cloning of hybrids with SPL and PBL (stimulated and non-stimulated)indicated that PBL could not be used for hybridoma formation. Cloningwas performed 4-6 weeks after fusion in 50% epithelial conditioned media(ECM) (pre-incubated for 24 hours at 37° C. in 96-well plates) and 50%RPMI 1640 containing 15% FCS. Results were determined at in 2-2.5 weeks.Cloning efficiency (1.5-2 cells per well) was 50-80% for SPL and 10-30%for PBL. ELISA using rabbit anti-human immunoglobulin and MAH-L,Hindicated that the total immunoglobulin production was present in 90-95%of growing hybrids with PBL and 80-85% with SPL hybridomas. Based on SPLwas selected for PWM stimulation and in vitro immunization.

In order to increase the efficiency of hybridization, splenocytes weretreated with 2.5 mM Leu-Ome and fused with B6B11 cells at ratio of 1:1or 1:2 (B6B11: SPL) (see Table 2). The effect on this treatment wasapparent after 18-24 hours of cultivation with PWM; SPL without Leu-Ometreatment exhibited blasts only after three days. The efficiency ofhybridization of Leu-Ome-treated SPL was somewhat higher (80%) comparedwith non-treated SPL (72%). This treatment considerably increased (93%)the number of Ig-secreting hybrids.

TABLE 2 Effect of Leu-Ome treatment of splenocytes on the efficiency oftheir hybridization with B6B11 cells (data from 3 spleens) Number Wellswith hybrid Wells² with Ig Lymphocytes of wells populations, (%)secretion, (%) SPL 1440 1034 (72) 825 (80) SPL-Leu-Ome  864  691 (80)642 (93) ¹Splenocytes were isolated in LSM. One portion was treated withLeu-Ome (2.5 mM, 40 minutes in serum-free RPMI 1640), the other servedas a control. Prior to fusion both portions were cultured for 7 days incomplete RPMI 1640 supplemented with 15% FCS in the presence of 5 μg/mlPWM. ²Ig production was determined by ELISA with mouse monoclonalantibodies to H- and L-chains of human Ig.

The heteromyeloma cells were fused with Leu-Ome-treated splenocytesimmunized with Salmonella Minnesota Re595 (Re595) in the presence of PWMand mouse thymocyte conditioned media (TCM) (Table 3). The hybridomaculture supernatants were tested for anti-bacterial antibodies atdifferent stages of hybrid growth: (1) after transferring respondingpopulations to 24-well plates and (2) after cloning and subsequentclonal expansion. Two independent clones producing anti-bacterialantibodies were selected. ELISA using immobilized lipoplysaccharide(LPS) or immobilized Re595 and LPS in solution determined that theantibodies produced by both clones reacted with LPS.

ELISA using immobilized Re595 monoclonal mouse anti-human isotypes andgoat anti-mouse peroxidase conjugate absorbed with human immunoglobulin,determined that the antibody isotype was IgM-kappa. Both clones wereadapted to serum free media (SFM) by gradual replacing of the growthmedium containing 10% FCS. The maximal density upon culturing in SFM wasapproximately 1.2×10⁶ cells/ml. SFM-adapted cells were cloned asdescribed above. The efficiency and cloning time were similar to thoseof the cells cultured in serum-supplemented RPMI 1640 medium.

TABLE 3 Fusion of in vitro immunized splenocytes¹ with B6B11 cells.Number of fusion 1 2 3 Number of wells 288 864 576 Wells with hybridpopulations, 193 734 472 (%) (69) (85) (82) Wells with ig secretion, 173675 420 (%) (90) (92) (89) Primary response² to Re595,  9 —  17 numberof wells (4.5) (3.6) Secondary response³,  2 —  16 number of wellsNumber of responding — —  2 populations after cloning ¹Splenocytes aftertreatment with Leu-Ome (2.5 mM, 40 min) were in vitro immunized with S.minnesota Re595 (10⁷-10¹⁰ cells/ml) in the presence of PWM (5 ug/ml) andTCM for 7-9 days. Fusions with B6B11 cells were done at ratios 1:1 and1:2 ²ELISA of hybridoma culture supernatants from 96-well plates (rabbitanti-human Ig). ³ELISA of hybridoma culture supernatants aftertransferring in 24-well plates (rabbit anti-human Ig).

DNA analysis. FIG. 4 illustrates the distribution of the DNA content byparental lines, B6B11 heteromyeloma and B6B11-splenocyte hybrid. The DNAof heteromyeloma cells consists of 78.7% of the total parental DNA. TheDNA content of B6B11-splenocyte hybrid cells is 3% greater than that ofB6B11 cells.

Discussion

A partner cell line for production of human monoclonal antibodies wasgenerated by somatic hybridization of mouse X63.Ag8.653 and human RPMI1640 myeloma cells. Adaptation to medium with 8-Ag, subsequent cloningand selection by hybridization efficiency led to a heterohybrid clonewhich was designated B6B11. Fusion between heterohybrid lines andlymphocytes gives essentially stable productive hybrids (Raison R L, etal., 1982). The mechanisms underlying this phenomenon are unknown. It issuggested that human chromosomes or their fragments retained in thepartner line after the first fusion modify the intracellular environmentin such a way that the human lymphocyte chromosomes or fragments afterthe second fusion are stabilized (Oestberg L, and Pursch E., 1983). Thelarge number of chromosomes, the presence of hybrid marker chromosomesand increased DNA content observed in the experiments described herein,confirmed the hybrid nature of B6B11 cells. The DNA content of B6B11-SPLhybrid cells was also increased. Immunocytochemical testing forintracellular heavy and light chains and ELISA testing forimmunoglobulin secretion demonstrated that B6B11 cells produce neitherimmunoglobulins nor heavy and light chains. Fusion of B6B11 with SPLresulted in more hybrids than fusion with PBL (30-50 per 10⁷ SPLcompound to 1-5 per ⁷10 PBL). Cloning efficiency with SPL was 50-80% ascompared to 10-30% with PBL. Thus SPL were the more preferable partnerfor fusion. The culture media was conditioned by endothelial cells;which was deemed crucial for viability and clonogeneity of the hybrids.In the case of B6B11-PBL hybrids, immunoglobulin secretion was detectedin up to 95% of the hybrids. To increase the yield ofimmunoglobulin-secreting hybrids after fusion with SPL (up to 93%)Leu-Ome was used. Almost all hybrids secreted antibodies of unknownspecificity. The antibody production by B6B11 hybrids was stable for atleast 10 months. The hybrids were readily adapted to serum-free media,thereby facilitating a ex-vivo antibody production.

Two antibody-producing clones (with probably similar specificity to LPSof S. minnesota Re595) were obtained after fusion of immunized SPL withB6B11 cells. As demonstrated herein, human-mouse heteromyeloma, B6B11,is useful for producing human monoclonal antibodies to various antigens.Proper in vitro sensitization of lymphocytes is also of criticalimportance for generating human antibodies.

Experimental Procedures

Cell Culture. 8-Azaguanine (8-Ag) resistant mouse myeloma X63.Ag8.653(653) cells were transfected with plasmid pBgl-neoR (Dr. A. Ibragimov)as described below. The myeloma cells were maintained in DMEM mediumsupplemented with 10% fetal calf serum (FCS), 4 mM L-glutamine, 1 mMSodium pyruvate, non-essential amino acids and vitamins (FlowLaboratories). Prior to fusion the cells were passaged 3 times in thepresence of 20 μg/ml 8-Ag (Sigma) and 500 μg/ml G-418 (Gibco).

Human myeloma cell line RPMI 8226 (8226) was cultured in RPMI 1640medium with above-mentioned supplements (regular RPMI 1640). The hybridheteromyeloma B6B11 was cultured either in regular RPMI 1640 with 10%FCS or in serum-free media which represented 1:1 mixture of Iscove'smodification of Dulbecco medium (IMDM) and HAM F-12 (Flow Laboratories)supplemented with bovine serum albumin fraction #5, 2 mg/ml, (BSA)(Sigma), bovine insulin, 5 μg/ml (Serva), human transferrin, 5 μg/ml(Sigma), progesterone, 6 ng/ml (Gibco), hydrocortisone, 60 ng/ml(Gibco). Hybridomas were adapted to this serum free medium (SFM) bygradual replacement of the growth medium containing 10% FCS. All cellswere cultured in a humidified atmosphere of 5.5% CO₂/94.5% air at 37° C.

Human peripheral blood lymphocytes (PBL) were isolated using lymphocytesseparation medium (LSM) (Flow Laboratories) as per manufacturerinstructions. Spleens collected at autopsy not later than 2 hours afterdeath (males aged 50-60 years old) were homogenized and splenocytes(SPL) were isolated in LSM.

Production of Geneticin (G-418) Resistant 653 Myeloma Cells. Cells werewashed in sterile phosphate buffered saline (PBS) without Ca⁺⁺ or Mg⁺⁺.pBgl-neoR Plasmid DNA linearized by BamH1 (constructed by P. Chumakov,Institute of Molecular Biology of the Academy of Sciences of the USSR,Moscow, USSR) was added to the cell suspension. Prior to adding the DNAto the cell suspension, the DNA was twice phenol extracted usingphenol-ether at 4° C., 96% ethanol precipitated and dried under sterileconditions.

Transfection was performed by electroporation at 4° C. using a unitconstructed by L. Chernomordik (Institute of Electrical Chemistry of theAcademy of Sciences of the USSR, Moscow, USSR). Approximately 4×10⁶653myeloma cells and 3.5 μg of plasmid DNA were combined in an 80 μlelectroporation chamber. The final concentration of DNA was 44 μg/ml).An electrical current impulse of 1.7 Kv/cm was pulsed through thechamber for 100 μsec. After resting for 10 minutes the cells weretransferred to 0.5 ml complete media in 16 mm² wells at 5×1³0 and 2×⁴10cells/well. After 36 hours, 0.5 ml of media containing 1 mg/ml ofGeneticin (G-418) was added to a final concentration of 0.5 mg/ml.Subsequently, 50% of the media volume was changed every 2 days for 12days.

Production of heteromyeloma. G-418-resistant 653 cells were mixed with8226 cells at a 1:1 ratio and pelleted. 50% (v/v) polyethylenglycol(PEG) 3350 (Sigma) was added (200-300 μl per 4-5×10⁷ cells) for 1 minwith constant stirring. Several portions of serum-free RPMI 1640(RPMI-S⁻) were added for 5 minutes (first 10 ml), 1 minute (10 ml), and1 minute (30 ml). Cells were pelletted resuspended in regular RPMI 1640with 20% FCS, hypoxanthine (1×10⁴ M), aminopterine (4×10⁷ M), thymidine(1,6×10⁵ M) (HAT, Flow Laboratories) and 500 μg/ml G-418 and seeded in96-well plates (Linbro) at a density of 10⁵ cells per well. At two weeksthe medium (½ volume) was replaced with medium containing hypoxanthine(2×10⁴ M), thymidine (3.2×10⁵ M) (HT, Flow laboratories) and G-418 (500μg/ml). The procedure was repeated after two weeks.

Production of human monoclonal antibodies. Fusion of B6B11 cells withhuman lymphocytes was accomplished by the above-described method withfollowing modifications. Lymphocytes were mixed with B6B11 at a 1:1 or a1:2 ratio, pelleted, washed with RPMI 1640-S- and incubated with PEG(600 μl per 10⁵ cells) for 3 minutes with constant stirring. Theportions of added RPMI-S- were as follows: 10 ml/10 minutes, 10 ml/10 5minutes, 10 ml/l minute. Cells were pelleted, re-suspended in regularRPMI supplemented with 15% FCS and seeded in 96-well plates (1.5×10⁵cells per well). HAT-medium was added after 24 hours. The growth medium(½ volume) was replaced with fresh HAT in 7-9 days. HAT-medium wasreplaced with HT-medium at 15-18 days.

Cloning. Parent heteromyeloma and hybridoma cells were cloned by thelimiting dilution method in medium conditioned by human umbilical oraortic endothelial cells (Antonov A S, et al., 1986) (gift from Dr. A.Antonov) (ECM). 100 μl/well was incubated in 96-well plates at 37° C.overnight. Cells were planted at approximately 1 to 2 cells per well.The culture medium was tested for antibodies at 2.5-3 weeks.

Immunization in vitro. Freshly isolated lymphocytes were resuspended inRPMI-S- containing 2.5 mM L-leucine methyl ester (Leu-OMe) (Borrebaeck,CAK, et al., 1987) to a final concentration of 10⁷ cells per ml. After40 minutes of incubation at room temperature, cells were washed 3 timeswith RPMI-S- and resuspended in regular RPMI 1640 supplemented with 15%FCS. Medium conditioned by mouse thymocytes (TCM) was used as a sourceof lymphokines (Reading C L., 1982). Pokeweed mitogen (PWM) (Flowlaboratories) to a final concentration 5 μg/ml, TCM (25%) and antigen indifferent concentrations were added to the cell suspension. The cellsuspension (4-6×10⁶ cell/ml) was transferred to flasks (30 ml/75 cm²flask). Fusion was performed after 7-9 days of cultivation. ConcanavalinA (ConA) (Flow 5-10 μg/ml), Phytohemagglutinin (PHA) (Flow, 5-10 μg/ml)and lipopolysaccharide (LPS) (SIGMA, 10-15 μg/ml) were used instead ofPWM. S. minnesota Re595 (gift of Dr. O. Luderitz, Max Plank Institutefur Immunologie, Feiburg, Germany) was used as an antigen. The bacteriawere grown in medium containing 16 g/l tryptic soy broth (TSB), Difco),16 g/l brain-heart infusion (BHI) (Difco) and 4 g/l yeast extract (YE)(DIFCO) for 18 hours at 37° C. with constant stirring and then heatinactivated. The antigen concentration varied from 10⁷⁻¹⁰ ¹⁰ cells/ml.

Determination of antibodies and non-specific Ig production. Enzymelinked immunoassay (ELISA) was used to test hybridoma supernatants forthe presence of antibodies against Salmonella minnesota Re595 and LPS.

Screening for mAbs reactive against bacteria. 96-well plates werecovered with glutaraldehyde (1%, 100 μl per well) for 2 hours at roomtemperature. The plates were washed with distilled water 3 times.Bacteria were resuspended in 50 mM ammonium carbonate buffer (pH 9.6)and transferred to plates (5×10⁷ cells in 100 μl per well), centrifugedat 780×g for 30 minutes and washed with distilled water 4 times. Thesupernatants tested (100 μl) were supplemented with 0.1% Tween 20(Fluka), put into bacteria-containing wells and incubated for 1 hour atroom temperature. The media was then removed and the wells were washedwith distilled water. Affinity purified rabbit anti-human immunoglobulinconjugated to alkaline phosphatase (RAH-AP), diluted in tris-bufferedsolution (TBS, 50 mM, pH 7.4), containing 0.1% Tween 20 was added to 1μg in 100 μl per well. After 1 hour of incubation at room temperatureand 6 washes with distilled water 100 μl of 4-nitrophenyl-phosphate (1mg/ml, Sigma) in diethanolamine buffer (10% diethanolamine, 0.5 mMMgcl2, pH 9.8) was added. After 1 hour, the results were read using aMultiscan (Flow Laboratories) at 405 nm. The negative control wasculture medium RPMI 1640 supplemented with 15% FCS.

Screening for mAbs reactive against lipopolysaccharide. LPS was purifiedfrom Salmonella Minnesota Re595 as described (Galanos G, et al., 1969).The LPS preparation was sonicated and transferred to the plates at 2.5μg per well in 5mM ammonium carbonate buffer (pH 9.6). After overnightincubation at room temperature, the above described procedures fordetermining mAb reactive against bacteria were performed.

Screening for non-specific production of mAbs. Non-specific productionof immunoglobulin and separate chains was assessed after the addition of100 μl of rabbit anti-human immunoglobulin (10 μg/ml in phosphatebuffer, PBS, pH 7.2) or 100 μl/well (10 ng/ml in PBS) of mousemonoclonal antibodies to light and heavy chains of human immunoglobulin(MAH-L, H) (Rokhlin O V, 1989) (gift of O. Rochlin, C R C, Moscow).Subsequent procedures were performed as described above.

Determination of the isotype of secreted antibodies. The isotype ofhuman antibodies was determined by ELISA using murine anti-human lightand heavy chains (MAH-L, H) and goat anti-mouse immunoglobulin (25ug/ml) conjugated to peroxidase and absorbed with human immunoglobulin.

Determination of cytoplasmic light or heavy chains production.Production of cytoplasmic light and/or heavy chains in hybridomas, B6B11and the parental cell lines was estimated immunocytochemically using theperoxidase-anti-peroxidase system (PAP). Cell smears were air-dried,fixed for 45 seconds with 10% formaldehyde (v/v) and 45% acetone (v/v)in phosphate buffered saline (PBS, 10 mM NaH₂PO₄, pH 6.6) and incubatedwith MAH-L,H (200 μl, 5-10 mg/ml). Then 1 ml rabbit anti-mouseimmunoglobulin (38 mg/ml in PBS) was added. All incubations were 30minutes. Washings were performed using PBS for 10 minutes.

Chromosomal analysis. Preparations of metaphase chromosomes wereobtained by the following technique. Colchicine was added to cellsduring exponential growth (1.5-2 hours to parental lines and B6B11cells). Cells were then trypsinized and stained for G-banding asdescribed (Seabright S., 1971) (10-15 plates from each line). To countchromosome number, at least 50 metaphase figures were analyzed for eachcell line.

DNA analysis by flow cytometry. To estimate the DNA content the cells(1×10⁶) were fixed with 1 ml 70% ethanol, washed, incubated for 2-3hours with 0.3 mg/ml Ribonuclease A (Serva) in Hank's solution (pH 7.4)and stained for 2 hours with propidium iodide (0.05 mg/ml, Sigma) inHank's solution. The DNA content was measured in a FACS-IIcytofluorometer (Becton Dickinson). Fluorescence was excited by an argonion laser at 488 nm (164-05 Model, Spectra-Physics) at a power of 400 mWand registered behind a 600 nm long pass interference filter (DitricOptica).

Parental lines. The myeloma line 653 was maintained in DMEM supplementedwith 10 FCS, 20 ng/ml 8-Azaguanine and 500 μg/ml G-418. The myeloma line8226 producing lambda chains of human Ig was cultured in RPMI-Ccontaining 10% FCS. In order to create a heteromyeloma, a non-producingclone of 8226 line was selected by cloning in ECM (2 cells per well).Lambda chain production was estimated at 2-2.5 weeks using MAH-L, H. Thefrequency of non-secreting clones was 1×10³.

EXAMPLE 2 Trioma MFP-2, a Fusion Partner for Generating Human MonoclonalAntibodies Introduction

A precursor hybridoma cell line was obtained by hybridization of thecommercially available human myeloma cell line RPMI 8226 and mousemyeloma X63.Ag8.653 resistant to both 8-Azaguanine (8-Ag) and Geneticin418 (G-418). One of the resulting clones, B6B11, was selected in thepresence of G-418. B6B11 was grown in the presence of increasedconcentrations of 8-Ag and is resistant to both G-418 and 8-Ag (SeeExample 1).

Although B6B11 can be used to make human hybridomas by fusing with humanlymph node-derived lymphocytes or spleen-derived lymphocytes, B6B11 wasnot capable of fusing with human peripheral blood lymphocytes (PBL) orresulted in a very low yield of hybrids (see example 1).

In order to overcome this problem, B6B11 was fused with human lymph nodelymphocytes and several hybrids were obtained. The resulting cells wereanalyzed for human immunoglobulin production or production of separateimmunoglobulin chains. Those clones, which did not synthesizeimmunoglobulin or immunoglobulin chains were selected for furtherevaluation in terms of fusion capability and antibody secretionpotential. These hybrids were determined to be quite stable. Thesefusion products were designated “modified fusion partner” (MFP) cells.These MFP cells as the product of the fusion of the B6B11 hybridoma andlymphocytes are referred to herein as “trioma” cells because they are,in essence, the product of a three fused cells. One of the clones,MFP-2, exhibited a very high efficiency for fusing with peripheral bloodlymphocytes as well as for fusing with human lymphocytes of any variedorigin (i.e. lymph nodes, spleen, Peyer's patches etc). MFP-2 wasselected on the basis of its superior characteristics and stability as afusion partner and was used in the experiments described herein below.

The products of fusions between the MFP trioma cells and lymphocytes arereferred to herein as “tetroma” cells becase they are, in essence, theproduct of four fused cells.

Results

Immunoglobulin Production. In order to demonstrate that human hybrioma(trioma) fusion partner cell line, MFP-2, is capable of fusing withhuman lymphocytes and producing high yields of hybrids with stableimmunoglobulin production, experiments were performed using humanlymphocytes from different sources.

The heteromyeloma cell line, B6B11 (precursor to MFP-2), can be fusedwith high efficiency with lymph node and spleen lymphocytes. (See,Example 1). Up to 90% of the resulting hybrids produced IgG or IgM.However, B6B11 was incapable of fusing to lymphocytes derived fromperipheral blood (PBLs). The trioma cell line, MFP-2, (resulting from afusion between B6B11 and human lymph node lymphocytes) overcame thisproblem and exhibited high fusion efficiency with PBL, yielding a highrate of immunoglobulin production by the resulting tetroma hybrids. Thecapability of MFP-2 to fuse with PBL was tested in two ways: (1) byfusion with freshly isolated lymphocytes in suspension, and (2) byfusion with thawed lymphocytes which had been stored frozen for variousperiods of time. (See Experimental Procedures). The results of theseexperiments are shown in FIG. 5.

The fusion efficiency was 10⁵ (1 hybrid per 10⁵ lymph-mphocytes). Thirtyprimary hybridoma (tetroma) populations were obtained and analyzed forcapacity to secrete immunoglobulin. (A primary hybridoma population islikely to be a mixture of two or more individual clones). Twenty-sevenpopulations (90%) produced IgM at a level 5-fold greater thanbackground. Twenty-four populations (80%) secreted IgE at a level 5-foldgreater than background. The fusion of MFP-2 with lymphocyte suspensionswhich had been frozen and thawed also resulted inimmunoglobulin-producing hybrids. Nineteen percent and 11% of thesehybridoma populations produced human IgM and IgG respectively. Theefficiency of fusion, itself, was not effected by the freeze-thawprocedure. These results demonstrate that both freshly isolated as wellas frozen PBLs can be used to generate human hybridomas capable ofproducing antibody.

Identification of tumor-associated antigens and production of specificantibodies using the MFP-2 fusion partner: Human monoclonal antibodiesagainst thyroglobulin. In this experiment, human anti-thyroglobulinantibodies were generated by MFP-2 fusion using lymph nodes frompatients diagnosed with thyroid adenocarcinoma. A periclavicular lymphnode was excised during lymphadenectomy surgery from a female thyroidcancer patient and lymphocytes were isolated and fused with MFP-2,generating tetroma cells.

The resulting hybridomas (tetromas) were tested for production of humanantibodies reactive against thyroglobulin using an enzyme linkedimmunoassay (ELISA) procedure. Purified human thyroglobulin was used tocoat a microtitre plate. Results are shown in FIG. 6. Thirty-three of144 tetromas exhibited a response against the thyroglobulin antigen.Eight of these were particularly strong. (See FIG. 6). Thus, lymphnode-derived tetromas from this thyroid cancer patient were producinganti-thyroglobulin antibodies. This was an unexpected and surprisingresult because the patient had no known history of autoimmune (i.e.anti-thyroid antibodies) disease. This suggests that the antibodiesproduced in this patient to thyroglobulin were induced by the presenceof cancerous thyroid adenocarcinoma cells. Cancerous thyroidadenocarcinoma cells are known to secrete thyroglobulin. This experimentdemonstrates that tumor cells can induce a humoral immune response totumor-associated antigens and that the antibody-producing cells can beidentified and immortalized through the techniques described hereinusing the MFP-2 fusion partner in order to produce human anti-tumormonoclonal antibodies.

Production of human monoclonal antibodies against breast cancerassociated antigens. In another experiment, human monoclonal antibodieswere produced against cancer associated antigens using lymph node andperipheral blood lymphocytes from breast cancer patients. Axillary lymphnodes were excised from breast cancer patients who underwent mastectomyor lumpectomy. Lymphocytes isolated from these lymph nodes were fused toMFP-2 and the resulting tetromas were screened against breast cancercell lines MCF7, SK-BR-3, ZR-75-1. Nearly all the tetromas wereproducing IgG or IgM (approximately 85% and 10% respectively).Surprisingly, nearly 15% of the tetromas assayed against breast cancercell lines produced antibodies specifically directed against cancercells. The tetroma supernatants were tested in two ways: (1) on a livecells in the CELISA (cellular ELISA) assay and (2) by Western blottingusing cell lysates. The molecular weight range of the specific antigensrecognized by human monoclonal antibodies was 25 to 160 kDA. In order todelineate the nature of the antigenic target, immunoprecipitationfollowed by microsequencing is performed. In addition, random peptidecombinatorial libraries are used to identify the molecular targets ofthe cancer-specific antibodies.

In one patient with Stage IV breast cancer, lymph nodes were notavailable so PBLs were fused to MFP-2 and 156 tetromas were obtained.The tetromas were analyzed for immunoglobulin production as well as forcancer-specific antibody production. IgM was produced by 28 tetromas; 87tetromas produced IgG. Four of the IgM antibodies and seven of the IgGantibodies were identified as reactive against cellular antigens; threeIgM anti-bodies and four IgG antibodies were specific for breast cancercells. The rest of the tetromas exhibited immunoreactivity against othercell types including human prostate cancer cell lines, human diploidfibroblasts and human skin fibroblasts. These latter antibodies wereprobably directed to common antigens (common for normal and cancerouscells).

The PBLs were isolated from the blood of a patient who received 77cycles of chemotherapy which would reasonably be expected to have adepressing effect on the patient's immune system. None-the-less, thispatient still produced anti-cancer antibodies suitable for fusing withMFP-2.

Human tetromas generated from fusing MFP-2 and prostate cancerlymphocytes are tested for the presence of PSA-specific antibodies aswell as antibodies directed to prostate cancer cell lines LNCaP, DU-145,and PC-3.

Production of human antibodies against infectious disease-associatedantigens. Infectious diseases are commonly accompanied by awell-developed humoral and cellular immune response. Patients withcertain infections often contain large numbers of specific antibodyproducing cells. One important application of the antibody immunotherapydescribed by the present invention, is the production of humanmonoclonal antibodies to proinflammatory cytokines which are involved inseptic shock. Among these targets are cytokines such as tumor necrosisfactor α (TNF-α) and interleukin-1a (IL-1a). Additional targets includeother cytokines and lymphokines, infectious agents and their toxins,including tetanus toxin, anthrax toxin, botulinum toxin, and lipid A.The peripheral blood of patients infected with bacteria, fungi, protozoaor viruses typically contains circulating antibody-producing cells whichcan be isolated and used as a source for fusion with MFP-2. For example,PBLs from patients with septic shock, Hanta virus infection, HIV,HTLV-I, HTLV-II, influenza, hepatitis, or herpes virus can be fused withMFP-2 and the resulting tetroma cells can be screened against therespective antigens. In AIDS, in particular, patient lymphocytes can beimmortalized using the techniques described herein in order to generatebulk quantities of anti-HIV antibodies for use in passive immunotherapyin an autologous or heterologous manner.

Production of human antibodies against autoimmune disease. A generalconsideration for the use of human monoclonal antibodies in autoimmunedisease is to block autoantibodies, or to block CD4⁺ T cells which areinvolved in autoimmune cellular cytotoxicity. In one approach, humanmonoclonal antibodies against CD4⁺ cells are generated following fusionwith the MFP-2 trioma cell. Resulting tetroma cells which produceanti-CD4 antibodies are used to reduce or deplete CD4⁺ T cells, therebyrelieving autoimmune cellular attack. In another approach MFP-2 is usedto generate tetroma cells capable of producing anti-idiotypic antibodiesdirected to specific autoantibodies. For example, autoimmune thyroiditisis an autoimmune dysfunction in which there is a high titer ofanti-thyroglobulin antibodies in a patient's plasma. PBL-derivedlymphocytes are isolated from such patients for fusion with MFP-2. Theresultant tetroma cells are screened for those capable of producingantibodies with a substantial anti-idiotypic immune response directedagainst the autoantibodies reactive with thyroglobulin. Theseanti-idiotypic antibodies are then used to modulate the autoimmunedisease by reducing or depleting the anti-thyroglobulin antibodies. Suchan approach may be used autologously or heterologously. In an autologousapproach, the anti-idiotypic antibody-producing cells are identified inperipheral blood of the patient to be treated, then isolated and fusedwith MFP-2 and following selection for specific anti-anti-thyroglobulinantibodies, passively administered to the original patient. In aheterologous approach, the anti-anti-thyroglobulin antibodies areadministered to a different patient.

Other Applications: Preventing rejection of transplanted organs, bloodclotting. Among other applications of human monoclonal antibodies, isprevention of organ transplant rejection by blocking T cells through theOKT-3 (anti-CD3) marker. Antibodies to adhesion molecules (anti-integrinantibodies) also prevent migration of immune cells, which is important,for example in rheumatoid arthritis. Blood clotting may be modulated,for example, in acute cardiac ischemia following coronary angioplasty,using human monoclonal antibodies against GPIIb/IIIa of platelet.Intravenous infusion of immunoglobulins helps to neutralize theFc-receptor mediated cell aggregation of platelet or other blood cells(e.g. thromobytopenic purpura).

In addition, this approach may be used to detoxify or neutralize toxinor venom exposure. Such exposures include, but are not limited to snake,spider or poison toad bites or yellow jacket or scorpion stings. Thehorse anti-serum currently used to neutralize rattle snake venom causesserum sickness disease in 30% of cases.

There is a shortage of natural human immunoglobulin required for thesekinds of treatments. The human monoclonal antibody production systemdescribed herein facilitates production, in vitro, of unlimitedquantities of human immunoglobulins which can be selected to fitparticular need. For example, in the case of immunoglobulin which blocksFc receptors, instead of treating the patient with the pooledpreparation of immunoglobulins where only a small fraction of moleculespossess the required qualities, the immunoglobulin preparation of themolecules with the required properties can be produced using the fusionpartner described herein.

Discussion

There has long been a need for human monoclonal antibodies fordiagnosis, treatment, and monitoring of cancer. Attempts to employxenoantibodies in clinical trials have not produced promising results.Non-human antibodies from mice, for example, cause development of ahuman anti-mouse immune response, sensitization to foreign protein whichmay eventually result in anaphylactic reaction, and lack of biologicaleffect since the effector properties of the xenoantibodies may mismatchthe components of the human immune system. Human monoclonal antibodieshave numerous advantages. One is that human monoclonal antibodies canidentify those tumor-associated antigens (TAA) which are immunogeniconly in humans, while xenoantibodies in most cases recognize thoseantigens and antigenic epitopes which express immunodominance in a hostand are often the tissue specific epitopes. Another advantage is thewell-developed interaction of human monoclonal antibodies with theeffector components (such as complement) of the host immune system. Inaddition, allergic and/or anaphylactic reaction to the injectible humanmonoclonal antibodies is less of a concern since human monoclonalantibodies are syngenic in human subjects. Alternative attempts havebeen made to develop antibodies such as chimeric antibodies (partiallyhuman, partially murine), where the Fc part of the murine immunoglobulinwas substituted with the human IgG-Fc. Humanized antibodies, are humanimmunoglobulins grafted with the CDR regions of the specific murineantibodies. Single chain (Fc) human antibodies have been developed inphage using phage display libraries. A downside of these approaches isthat the resulting antibodies are not natural; they have not emerged aspart of a natural immune response to cancer or infectious agent.

Use of the hybridoma techniques described herein and the availability ofthe MFP-2 trioma fusion partner cell line described herein, facilitatesidentification, immortalization, and ex-vivo expansion ofantibody-producing cells which emerge in vivo as a result of naturalhumoral immune responses to an antigen. Since such cells are a part ofthe natural immune system response, the antibodies produced by thesecells dovetail with the other components of the immune system and areable to provide an effective and specific biological response.

A number of breast cancer specific antigens have been described whichare potential targets for the immunotherapy of cancer, includingHER2/neu, Mucin 1 and Mucin 2, p53, c-myc, blood antigens T, Tn andsialyl-Tn, tuncated form of EGF, Lewis-Y antigen and others. Thepresence of circulating antibodies to these antigens have also beendescribed in cancer patients. (G. Moller, 1995). Lymph nodes areimportant sites of such antibody-producing cells. By isolating lymphnode (or peripheral blood) lymphocytes and immortalizing them by fusingthem with human hybridoma fusion partner MFP-2, hybrids (tetromas),which produce antibodies directed against cancer-associated antigens maybe obtained. As described above, specific monoclonal antibody producingcells are identified and may be produced in unrestricted fashion,ex-vivo (using bioreactors, SCID mice, etc). The antibodies may be usedtherapuetically as passive immunotherapy either autologously in the samesubject or heterologously in a different subject. Even another cancermay be treated, provided there is an overlapping tumor antigen.

Syngenic or allogenic use of human monoclonal antibody can be highlyeffective since such an antibody can be infused many times without therisk or threat of developing an anti-xenogenic immune response. Theinfused antibodies, depending on their effector functions, caninitialize complement dependent cytolysis of the target tumor cells, orantibody-dependent cellular cytotoxicity antibody dependent cellularcytotoxicity (ADCC) (by NK or CTL cells), or provide direct cytotoxiceffect through apoptosis.

Summary

A unique fusion partner cell line, MFP, was obtained which can be usedto generate specific human monoclonal antibodies. These monoclonalantibodies may be in vivo based on a natural immune response toinfectious agents, cancer cells or an autoimmune dysfunction, or can bein vitro based by immunization of human lymphoid cells in vitro.

The methods described herein for generating specific monoclonalantibodies may be used to provide adoptive humoral immunotherapy eitheras an autologous procedure or as a heterologous procedure. Lymphocytesisolated from a patient with a cancer or infectious disease areimmortalized by fusion with MFP-2. The resulting tetromas, producingantibodies directed to the respective antigens, are selected in vitro.Following selection, these antibody-producing cells are expanded andantibodies may be produced using a bioreactor or immune-deficient mice(e.g., nude mice or SCID mice). Such antibodies may then be used for thetreatment of the original donor as an autologous adoptive immunotherapyprocedure or for the treatment of a different subject as a heterologous,adoptive immunology procedure.

The developed antibodies may also be applied both to invasivediagnostics (imaging, immunoscintigraphy) or therapy (drug targeting,radioimmunotherapy, complement-dependent cytolysis, ADCC, apoptoticcytolysis etc.)

This approach also provides a method for identification of novel tumormarkers or novel infectious agent antigens. The immune system respondsto cancer cells or infectious agents by producing antibodies directed todifferent components of the foreign formation and can recognizedifferent neo-epitopes. Fusing tumor reactive or infectious agentantigen reactive immunoglobulin with MFP-2 can be used to identify noveltumor markers or infectious antigens. Such antibodies are important intreatment against specific cancers or infectious agents, and in thegeneration of specific imaging and diagnostic techniques. Previousattempts to generate human anti-tumor or anti-infectious antibodiesrequired forced or artificial immunization of a subject with purified orisolated antigen. In the present invention, the antigen may be unknown;the starting material for developing antibodies is the pool ofimmunocompetent lymphocytes which evolved as a part of natural immuneresponse to the foreign antigens presented in their natural form and innatural environment in vivo. In an autologous application, selection canbe conducted using an autologous tissue of interest (e.g. tumorbiopsies) which will increase the chances to select the right antibody.Also, autologous blood plasma and white blood cells can be used toselect for cytotoxic antibodies from the same donor.

Thus, the MFP fusion partner (1) allows fusion with peripheral bloodlymphocytes yielding high levels of hybrids; (2) allows consideration ofan adoptive humoral immunotherapy on an individual basis (selection ofthe antibodies against tumor cells or infectious agents derived from thesame donor the lymphocytes were obtained from and the autologoustreatment of the patient); (3) fusion with the donor's lymphocytesundergoing immunization in vitro; (4) allows use of frozen lymphocytesor lymphocytes derived from plasmapheresis as a source ofantibody-producing cells.

Experimental Procedures

Hybridoma fusion partner MFP-2 was developed as a trioma cell line byfusing non-producing heteromyeloma B6B11 with human lymphocytes isolatedfrom the paraclavicular lymph node.

Isolation of lymphocytes. Paraclavicular lymph nodes from a patientdiagnosed with metastatic thyroid cancer were excised during the surgeryand placed into sterile conservation media RPMI1640 supplemented withL-glutamine (4 mM), non essential amino acids (100×stock), vitamins(100×stock), sodium pyruvate (1 mM) and Gentamicin (2×concentration).Lymph node tissue was transferred to a 100 mm tissue culture TC dish inthe same media and gently disrupted with forceps and scissors. Thedisrupted tissue was passed through a metal sieve (50 mesh) using aglass pestle. The suspension was transferred into 15 ml sterile conicaltubes containing lymphocyte separation media (Histopaque 1.077 Sigma) asan underlying layer at a ratio of 2:1 (lymphocytes suspension:Histopaque). Following centrifugation at 400×g for 20 minutes, an opaquering formed at the border between layers. Red blood cells (RBC) werepresent as a pellet at the bottom of the tube. If RBC are not present inthe starting lymphocyte suspension (which is a quite normal situationfor lymph nodes) the separation step can be skipped. The opaque ringcontaining lymphocytes was carefully collected using a Pasteur pipetteand was diluted 10-fold diluted with regular serum-free RPMI 1640. Cellswere spun at 300×g for 10 minutes and washed twice with media.

The final lymphocyte suspension was diluted with media and cells werecounted using 0.05% Trypan Blue. Cell viability after isolation wasusually 95%. Total yield was approximately 4×10⁷ cells.

Preparation of B6B11. Heteromyeloma B6B11 was grown in RPMI 1640 with10% cosmic calf serum (Hyclone), standard set of supplements (L-Glu, 4mM non-essential amino acids, vitamins, Sodium Pyruvate) withoutantibiotics. Before fusion, cells were cultured in the presence of 8-Ag(20 μg/ml) to avoid reversion of HAT-sensitive cells to wildtype. Cellswere grown to a density of 10% in logarithmic growth phase.

Cell fusion. Both B6B11 cells and lymph node lymphocytes were washed 3times by centrifugation at 300×g for 5 minutes in order to remove anyresidential protein in the media. Cells were mixed at a ratio of 5:1(lymphocyte: myeloma) and spun at 300×g for 10 minutes. The supernatantwas carefully and completely removed the pellet was “puffed” gently and100 μl of PEG/DMSO solution warmed to room temperature was added to thecell mixture which was gently tapped for 3 minutes. Then 15 ml of Hank'sBalanced Salt Solution (HBSS) and PBS (1:1)(from a 10×stock, Cellgro)were added as follows: 10 ml slowly in 10 minutes, then 5 ml over 5minutes, then 10 ml of complete media (media for cell culturing) over 5minutes and finally 5 ml over 1 minute. The total volume was 30 ml. Then600 μl of HT solution (of 10×stock) and 1 drop (about 20-30 μl) of DMSOwere added to the tube. The cell suspension was mixed in a tube,transferred to Petri dish (100×15) and incubated in a 37° C. CO₂incubator overnight. The cells were then harvested, pelleted at 300×gfor 10 minutes and resuspended in complete media supplemented withHAT-solution and HT-solution (both from 50×stock) and then plated into96-well plates in a 200 μl volume at about 250,000 cells per well. Twicea week, 50% of the media was replaced with fresh media. Cells werecultured in the presence of HAT and HT for 14-20 days before screeningfor antibody production.

ELISA screening for nonspecific immunoglobulin. ELISA plates were coatedwith polyclonal goat-anti-human IgG (Fc-specific) (Sigma),goat-anti-human IgM (μ-specific) (Sigma) or goat-anti-human Ig(G+M+A)H-chains (Sigma) in 100 μl of plating buffer (0.1 M Sodium Carbonate, pH9.0) at 100 ng per well. The plates were sealed with Parafilm or sealingcovers and incubated overnight at 4° C. The antigen was washed out withdistilled water twice. Residual drops of water were removed and 200 μlof blocking solution (0.4% dry non-fat milk in PBS) was added to thewells. Complete cell culture media served as a negative control. Humanserum (1:2000) was used as a positive control. Plates were incubated for2 hours at room temperature or overnight at 4° C. The plates were washed4 times with distilled water and secondary antibodies (same as captureantibodies but conjugated to HRP) diluted in 0.4% milk/PBS at 1:2000were added to the wells. After 1 hour incubation at room temperature thewells were washed 4 times with H₂O and peroxidase substrate(ortophenylendiamine in phosphate-citrate buffer with peroxide) wasadded to the plates. The color reaction was stopped by adding 20 μl of10% sulfuric acid. Colorimetric reading was performed on a Multiscanreader at A₄₉₂. Samples which exhibited at least a 3-fold increase overbackground were considered to be immunoglobulin-producing cells.

Assay for the intracellular (non-secreted) presence of immunoglobulinsor their individual chains. Cells which did not secrete immunoglobulinin the supernatant culture media were tested for the presence ofintracellular immunoglobulin-immunoreactive material. ELISA plates werecoated with goat-anti-human kappa chain (Sigma), goat-anti-human lambdachain (Sigma) and goat-anti-human IgH (G,M,A) as described above. Cellswere grown in 75 cm² flasks to the density 10⁶ cells per ml, harvestedand washed 3 times with HBSS. Cells were resuspended in PBS anddisrupted by sonication (8×15 seconds at 25 MHz on ice). The suspensionwas spun for 15 minutes at 10,000×g and the supernatant was used forimmunoglobulin testing. An equivalent of 2×10⁶ cells was used. As anegative control mouse fibroblasts 3T3 were used at the same proteinamount equivalent. The rest of the protocol was the same as describedabove for the hybridoma supernatant testing. Clones which showed thesignal equal to the control cells or lower were chosen as potentialcandidates for fusion with human peripheral blood lymphocytes. Thesetrioma clones were designated as modified fusion partner series (MFP-S)and numbered sequentially (MFP-1, MFP-2, MFP-3, etc.) Six non-producing,non-secreting triomas were selected for further analysis.

Selection for 8-Ag resistant MFP mutants. To use MFP trioma cells asfusion partners, the MFP cells were placed in complete media containingan increasing amounts of 8-Ag. Resistance to 8-Ag is determined by theimpaired enzyme HGPRT or its absence. Selection was therefore focused oncells which survived in the presence of 8-Ag. After 5 to 10 passages atthe lower concentrations of 8-Ag (5 μg/ml) the survivors were culturedin media with a higher concentration (10 μg/ml). This was repeated untila concentration of 20 μg/ml was reached. After 5-6 passages in thepresence of 8-Ag (20 μg/ml) cells were tested for their viability inHAT-media. None of the cells grown on 8-Ag survived after 3 days ofculture in the presence of HAT.

Fusion efficiency. The MFP clones were tested for ability to fuse withlymph node lymphocytes and PBL. MFP-2 yielded approximately 2-3 hybridsper 10⁵ lymph node lymphocytes and 0.7-1.5 hybrids per 10⁵ of PBL. Theimmunoglobulin secretion rate for the hybrids developed using MFP-2ranged between 0.5 to 15 ug/ml with no decrease over 7 months.

REFERENCES

Kohler G, and Milstein C., Nature 1975; 256:495

Levy, R., and Miller R A. Federation Proceedings 1983; 42:2650.

Posner M R, et al., Hybridoma 1983; 2:369.

Kozbor D, and Roder J., J.Immunology 1981; 127:1275.

Casual O, Science 1986; 234:476.

Glassy M C, Proc.Natl.Acad.Sci (USA) 1983; 80:6327.

Ollson L, et al., J.Immunol.Methods 1983; 61:17

Nilsson K. and Ponten J., Int.J.Cancer 1975; 15:321

Goldman-Leikin R E, J.Lab.Clin.Med. 1989: 113:335.

Brodin T, J.Immunol.Meth. 1983; 60:1.

Teng N N H, Proc.Natl.Acad.Sci. (USA) 1983; 80:7308.

Weiss M C, and Green H. Proc.Natl.Acad.Sci. (USA) 1967; 58:1104.

Oestberg L, and Pursch E., Hybridoma 1983; 2:361

Kozbor D, et. al., J.Immunology 1984; 133:3001

Shnyra A A, et al., In: Friedman H, Klein T W, Nakano M, Nowotny A, andEds. Advances in Exp. Medicine & Biology Endotoxin New York: Plenum,1990; 256:681.

Antonov A S, et al., Atherosclerosis 1986; 59:1.

Borrebaeck C A K, et al., Biochem.Biophys.Res.Commun. 1987; 148:941.

Reading C L., J.Immunol. Meth. 1982; 53:261.

Galanos G, et al., Eur.J.Biochem 1969; 9:245.

Rokhlin O V, 8th Int. Congress of Immunology, Berlin. Abstracts 1989; 6.

Seabright S., Lancet 1971; 2:971.

Yunis J J., Cancer Genetics and Cytogenetics 1980; 2:221.

Raison R L, et al., J.Exp.Medicine 1982; 156:1380.

Moller, G, 1995. (editor) Immunological Reviews Vol 145: TumorImmunology.

What is claimed is:
 1. A trioma cell which does not produce any antibodyobtained by fusing a heteromyeloma cell which does not produce anyantibody with a human lymphoid cell, wherein the heteromyeloma cell isdesignated B6B11 (ATCC Designation number HB-12481).
 2. The trioma cellof claim 1, wherein the human lymphoid cell is a myeloma cell.
 3. Thetrioma cell of claim 1, wherein the human lymphoid cell is a splenocyteor a lymph node cell.
 4. The trioma cell of claim 1, wherein the triomacell is designated MFP-2 (ATCC Designation number HB-12482).
 5. Atetroma cell capable of producing a monoclonal antibody having specificbinding affinity for an antigen obtained by fusing the trioma cell ofclaim 1 with a human lymphoid cell capable of producing antibody havingspecific binding affinity for the antigen.
 6. The tetroma cell of claim5, wherein the human lymphoid cell is selected from the group consistingof a peripheral blood lymphocyte, a splenocyte, a lymph node cell, a Bcell, a T cell, a tonsil gland lymphocyte, a monocyte, a macrophage, anerythroblastoid cell and a Peyer's patch cell.
 7. The tetroma cell ofclaim 5, wherein the antigen is selected from the group consisting of atumor-associated antigen, a cell specific antigen, a tissue-specificantigen, an enzyme, a nucleic acid, an immunoglobulin, a toxin, a viralantigen, a bacterial antigen and a eukaryotic antigen.
 8. A trioma cellgenerated by a method comprising: (a) fusing a heteromyeloma cell whichdoes not produce antibody with a human lymphoid cell thereby forming atrioma cell; (b) incubating the trioma cell formed in step (a) underconditions permissive to the production of antibody by the trioma cell;and (c) selecting a trioma cell that does not produce antibody, whereinthe heteromyeloma cell of step (a) is designated B6B11 (ATCC Designationnumber HB-12481).
 9. A tetroma cell generated by a method comprising:(a) fusing the trioma cell of claim 1 with a human lymphoid cell therebyforming a tetroma cell; (b) incubating the tetroma cell formed in step(a) under conditions permissive to the production of antibody by thetetroma cell; and (c) selecting a tetroma cell capable of producing amonoclonal antibody.